Methods for removing cytokines from blood with surface immobilized polysaccharides

ABSTRACT

The present invention is directed to a method for removing cytokines and/or pathogens from blood or blood serum (blood) by contacting the blood with a solid, essentially non microporous substrate which has been surface treated with heparin, heparan sulfate and/or other molecules or chemical groups (the adsorbent media or media) having a binding affinity for the cytokine or pathogen(s) to be removed (the adsorbates), and wherein the size of the interstitial channels within said media is balanced with the amount of media surface area and the surface concentration of binding sites on the media in order to provide adequate adsorptive capacity while also allowing relatively high flow rates of blood through the adsorbent media.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/261,390, filed Apr. 24, 2014, allowed, which application is adivisional of U.S. application Ser. No. 12/958,355, filed Dec. 1, 2010,now U.S. Pat. No. 8,758,286, which application claims priority to U.S.Application No. 61/265,675, filed Dec. 1, 2009. The teachings all ofwhich are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention is directed to a method for removing cytokinesand/or pathogens from blood or blood serum (blood) by contacting theblood with a solid, essentially non microporous substrate which has beensurface treated with a polysaccharide adsorbent, such as heparin,heparan sulfate and/or other molecules or chemical groups (the adsorbentmedia or media) having a binding affinity for the cytokine orpathogen(s) to be removed (the adsorbates), and wherein the size of theinterstitial channels within said media is balanced with the amount ofmedia surface area and the surface concentration of binding sites on themedia in order to provide adequate adsorptive capacity while alsoallowing relatively high flow rates of blood through the adsorbentmedia. The result is that transport of adsorbates to the binding siteson the media occurs largely by forced convection. By (forced) convectionis meant, for example, flow produced by a pressure gradient generated bya pump, by the application of external pressure to a flexible container(or internal pressure to a rigid container), by a gravity head/elevationdifference, or by the difference in arterial pressure and venouspressure in the patient being treated. The invention providesclinically-relevant adsorbtive capacity within the range of safe flowrates typically used in clinical extracorporeal blood circuits, e.g., indialysis, cardiopulmonary bypass, and extra corporeal membraneoxygenation of blood. The method is in direct contrast to the muchslower diffusive transport of adsorbates typically required with porousadsorbent media, which require adsorbates to diffuse through amicroporous membrane, and/or into microscopic pores before binding toadsorption sites on, behind, or within the media, and which thereforerequire very low flow rates to achieve significant separations duringeach passage of blood. The present invention also provides a method oftreating a disease by removing cytokines and/or pathogens from blood bycontacting blood with an essentially nonporous substrate coated with apolysaccharaide adsorbent, such as heparin, heparan sulphate, and/or,other adsorbent materials, and a device for performing the method andtreatment.

BACKGROUND OF THE INVENTION

A wide variety of disease conditions are characterized by the presenceof elevated concentrations of cytokines and/or pathogens in the bloodstream. Some such conditions are treated by therapies designed to killthe pathogen, e.g. through the administration of drugs, e.g.,anti-infective pharmaceuticals. Some other conditions are treated bytherapies that attempt to reduce the concentration of blood-bornecytokines or pathogens in the patient. Other diseases are treated bytherapies that attempt to directly remove only specific components fromthe patient's blood.

For example, Guillian-Barre syndrome is currently understood to be anautoimmune disorder triggered by viral infection that stimulates thebody's immune system to over produce antibodies or other proteins whichcan attack the patient's nervous system, causing increasing levels ofparalysis. Most patients recover over time, though such patients appearto be susceptible to recurrance of the condition from subsequent viralinfections. One method for treating Guillian-Barre syndrome involvesplasmapheresis to ‘clean’ the patient's blood by removing antibodiesbelieved to be attacking the patient's nervous system.

Certain biologically active carbohydrates and polysaccharides can removeharmful substances from blood and biological fluids.

Heparin is a polysaccharide that can be isolated from mammalian tissue.It has a very specific distribution in mammalian tissue; being presentonly in the basophilic granules of mast cells. Since its discovery in1916 by the American scientist McLean, heparin has been recognized forits ability to prevent blood from clotting, and for its relatively shorthalf-life in the body. Systemic heparin, administered by injection ofthe free drug, has been used clinically for more than 50 years as a safeand effective blood anticoagulant and antithrombotic agent. The effectsof heparin on blood coagulation/clotting diminish fairly quickly afteradministration is halted, making its use during surgery and otherprocedures effective and safe. That is, heparin's anticoagulant andantithrombogenic properties are useful during many medical procedures,for example to minimize undesirable interactions between blood and theman-made surfaces of extracorporeal circuits. Once the procedure isover, the administration of heparin may then be terminated. The heparinconcentration in the patient's blood diminishes to a safe level within afew hours because of its short half life in the body. This isparticularly important following surgery when healing depends on theability of blood to clot at the surgical site to avoid bleedingcomplications. In addition to its well established and continuing use inthe treatment of thromboembolic disorders, and the prevention ofsurface-induced thrombogenesis, heparin has more recently been found tohave a wide range of other functions apparently unrelated to itsfunction as an anticoagulant. For example, a large number of proteins inblood are now known to bind with high affinity, to heparin and/or theclosely-related polysaccharide heparan sulfate which is also found inanimal tissue, including the blood-contacting luminal surface of healthyblood vessels (where it may contribute to preventing circulating bloodfrom clotting on contact with the walls of the blood vessels). Someexamples are antithrombin (AT), fibronectin, vitronectin, growth factors(e.g. the fibroblast growth factors, the insulin like growth factors,etc.). Human serum albumin (HSA) also binds to heparin, but with a loweraffinity despite its high concentration in blood.

Others have considered utilizing the selective adsorption properties ofsystemic, free heparin for hindering infections, by introducing heparinfragments and/or so-called sialic-containing fragments directly into thevascular system. This therapy was based on the assumption that thesefragments would bind to the lectins on the microbes and block them sothey could not bind to the receptors on the mammalian cell surface.Although this approach has been investigated by many scientists, onlylimited success has been reported to date. The most common problem hasbeen bleeding complications associated with the large amounts of freeheparin introduced into the blood stream, e.g., by injection, to achievea clinically-useful reduction of pathogenic microbes. The presentinvention does not require the use of any free, systemic heparin forefficacy, and thus may eliminate bleeding complications. This isaccomplished by permanently binding the heparin or heparan sulphate to asolid substrate with high surface area, and exposing it to the bloodwithin a cartridge or filter containing this adsorption media.

The following references deal with issues discussed above:

Weber et al. (Weber V, Linsberger I, Ettenauer M, Loth F, et al.Development of specific adsorbents for human tumor necrosisfactor-alpha: influence of antibody immobilization on performance andbiocompatibility. Biomacromolecules 2005; 6: 1864-1870) reportedsignificant in vitro binding of TNF using cellulose micro particlescoated with a monoclonal anti-TNF antibody, while Haase et al. (Haase M,Bellomo R, Baldwin I, Haase-Fielitz A, et al. The effect of threedifferent miniaturized blood purification devices on plasma cytokineconcentration in an ex vivo model of endotoxinemia. Int J Artif Organs2008; 31: 722-729) reported a significant reduction in IL-1ra, but notin IL-6, using a similar ex vivo methodology as ours but with a porousadsorption device. In vivo, Mariano et al. (Mariano F, Fonsato V,Lanfranco G, Pohlmeier R, et al. Tailoring high-cut-off membranes andfeasible application in sepsis-associated acute renal failure: in vitrostudies. Nephrol Dial Transplant 2005; 20: 1116-1126) are able tosignificantly reduce several circulating cytokines with hemoperfusionand a high cut-off polysulphone membrane, but also reported a loss ofserum albumin. The putative clinical relevance of these findings aredemonstrated by Schefold et al. (Schefold J C, von Haehling S, CorsepiusM, Pohle C, et al. A novel selective extracorporeal intervention insepsis: immunoadsorption of endotoxin, interleukin 6, andcomplement-activating product 5a. Shock 2007; 28: 418-425) who in arandomized study of 33 patients with septic shock are able tosimultaneously reduce circulating endotoxin, IL-6, and C5a levels byselective immunoadsorption, resulting in improved organ function.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for theremoval of cytokines and/or pathogens from mammalian blood by contactingblood with a solid, essentially nonporous substrate coated withselective adsorbent molecules, biomolecules or chemical groups. Suchselectively adsorbent molecules may include polysaccharides, such asheparin, heparan sulphate, polyethylene imine (PEI), sialic acid,hyaluronic acid, and carbohydrates with mannose sequences. When usedprophylactically, e.g. during the collection or transfusion of bankedblood, or in direct patient-to-patient transfusion of blood, the use ofthe present invention can also be used to lessen or eliminate the spreadof disease. Thus the present invention may be used both to preventdisease, and to help cure it in previously infected patients.

One object of the invention is to provide a therapy for treating anexisting disease by removing cytokines and/or pathogens from mammalianblood by contacting mammalian blood with a solid essentially nonporoussubstrate coated with heparin and/or other adsorbent molecules andreturning the blood to the patient suffering from the disease.

The above mentioned objects are not intended to limit the scope of theinvention in any way.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of TNF-α binding to heparinized beads.

FIG. 2A is a graph of the amount of TNF-α adsorbed on the heparinizedcrystals.

FIG. 2B is a graph of the amount of TNF-α adsorbed onto controlcrystals.

DETAILED DESCRIPTION 1. Removal of Cytokines or Pathogens from the Blood

A first aspect of the present invention provides a method for theremoval of cytokines and/or pathogens from blood, such as mammalianblood, by contacting the blood with a solid substrate e.g., coated withheparin and/or other adsorbent carbohydrates and/or polysaccharides.

In an embodiment of this method, heparin is immobilized onto the surfaceof the substrate. The inventors have found that immobilized heparinbound to a surface is effective for removing a significant amount ofcytokines and pathogens from blood. However, the flow rates typical ofextracorporeal blood circuits require that the adsorbent ‘bed’ bedesigned to allow relatively high flow rates to operate safely. This isin part due to the universal tendency of slow-moving or stagnant bloodto form dangerous clots. In the present invention the substrate isdesigned with sufficiently large interstitial dimensions to permit ahigh flow rate of blood over the substrate without a large pressuredrop. That is, as blood is taken from a mammalian patient, it is passedover the substrate at a flow rate whereby the delivery of adsorbates tothe surface of the adsorbent bed is characterized primarily by forcedconvection. This is in contrast to the much slower process of moleculardiffusion that occurs in the use of highly porous adsorbent media (e.g.porous silica, sephadex, crosslinked polystyrene and other sizeexclusion media), and many other microporous media. Molecular diffusionis also required when selectively-permeable barrier membranes are usedtogether with adsorption media, e.g., to prevent contact of theadsorption media by blood cells and/or high molecular weight solutesduring affinity therapy.

The binding of cytokines and pathogens by heparin and/or other adsorbentmolecules during convection transport is particularly effective underthe relatively high-flow conditions typically employed in the (safe)operation of extracorporeal blood circuits, e.g. when measured by linearflow velocity, ≥8 cm/min, preferably about ≥24 cm/min, and morepreferably about 24-329 cm/minute, or, when measured by flow rate,around >50 mL/minute and preferably >150 mL/minute but less than about2000 mL/minute. Adsorption within the pores of microporous media, incontrast, may require much lower flow rates through adsorption beds ofpractical size in order to achieve an adequate separation orpurification, i.e. <50 mL/min to as low as <1 mL/min!

It is recognized that, strictly speaking, it is ‘residence time’ on theadsorption column that needs to be much longer for a media requiringdiffusive transport of adsorbates to the adsorbent site within themedia, when compared to the lower residence time needed to convey anadsorbate to the binding site (on an essentially nonporous media) byforced convection. However, there are practical limits to the dimensionsof a safe and effective adsorbent cartridge, column, filter, etc.,especially with respect to the maximum hold-up volume of blood it cancontain, and the flow velocity of blood or serum past the adsorptionmedia. For this reason average flow rate through the adsorption deviceis considered to be an important design variable.

Convection kinetics and diffusion kinetics can be compared in theremoval of cytokines or pathogens from flowing blood: Adsorption mediathat depend on diffusion transport generally use very porous materialswith extremely high internal surface area due to the presence ofmicroscopic pores. Media suited for convection transport, on the otherhand, generally rely on macroscopic “channels” or visible intersticesbetween solid, essential nonporous material, such as particles, beads,fibers, reticulated foams, or optionally spiral-wound dense membranes.

Media that rely on forced convection transport are generally moresuitable for high-flow rates, while media that rely on the much slowerdiffusion transport are much less effective when high flow rates andshorter residence times are required. For this reason, in anextracorporeal blood purification device, an adsorption media that doesnot require the adsorbate to slowly diffuse into pores within theadsorbent media is much preferred. When blood is pumped through circuitsfabricated from man-made materials it is a general practice to employrelatively high blood flow rates in order to prevent stagnation andreduce the risk of clotting. On the other hand, extremely high flowrates must be avoided because they can expose blood cells to high shearrates and impingement damage that can rupture or otherwise damage bloodcells. The present invention, therefore, provides a method and devicefor removing cytokines and/or pathogens from blood using the preferredcharacteristics of convection transport and its desirable, more-rapidkinetics. This is achieved by passing/flowing blood over an essentiallynon-microporous substrate that has been surface treated with adsorbentmolecules, e.g. heparin, and which is therefore capable of binding thedesired cytokine or pathogens to remove them from the blood. It is alsopossible to use a microporous substrate in the present invention ifsurface treatment renders that substrate effectively nonporous. This canoccur intentionally or inadvertently, when surface treatments duringmedia manufacturing block the pores. This converts the microporoussubstrate to one that does not require diffusion of adsorbate into poresto bind to the media.

The claimed methods are intended to be applied primarily inextracorporeal therapies or procedures, although implantable devices arealso possible “Extracorporeal therapies” means procedures that areconducted outside the body, such as therapies in which desired productslike oxygen, blood-anticoagulants, anesthetics etc can be added to bodyfluids. Conversely, undesired products like naturally occurring toxinsor poisons can be also removed from body fluids with specific types ofextracorporeal circuits. Examples are haemodialysis and haemofiltrationwhich represent technologies whereby blood is depleted of wasteproducts. Adsorption on activated carbon has been used to removeblood-borne poisons, and so forth.

Whole blood and blood serum from mammals can be used in the presentinvention. The amount of blood or blood serum that can be used in theclaimed methods is not intended to be limited. It can range from lessthan 1 mL to above 1 L, up to and including the entire blood volume ofthe patient when continuous recirculation back to the patient isemployed. One or more ‘passes’ through the adsorption bed may be used ifneeded. The blood may be human or animal blood.

Adsorption media to remove cytokines or pathogens from blood areoptimized according to the present invention for use in traditionalextracorporeal blood circulation with flow rates >50 mL/min, andpreferably between about 150 and 2000 mL/min. If measured by linear flowvelocity, ≥8 cm/min, preferably about ≥24 cm/min and more preferablyabout 24-329 cm/min. Such high flow rates create short residence timeswithin the adsorption column and convection transport dominates overBrownian diffusive transport. This is particularly important for bindinglarge MW proteins or cytokines such as TNF-α and larger particles suchas viruses, bacteria and parasites because they diffuse very, veryslowly. In the present invention the dominant adsorption sites availablefor removing cytokines and pathogens lie at the surfaces within theinterstices of the media bed through which the blood flows or isdelivered by forced convection. To treat blood, the interstitialchannels need to be large enough to allow the transport of red bloodcells, which are an average 6 microns in diameter. To allow a packedadsorption cartridge to be placed into an extracorporeal circuit withhigh blood flow rate, the interstitial channels must be several timeslarger than the diameter of red blood cells. This can prevent high shearrates that lead to hemolysis while simultaneously minimizing pressuredrop in the blood that flows through the packed bed or cartridge.Additionally, the media is preferably rigid to minimize deformation thatcould clog the filter cartridge by compaction. Based on thesepreferences, an optimized rigid media balances interstitial channel sizeand total surface area, e.g., for efficient removal of pathogens and/orcytokines in high-flow extracorporeal blood circuits.

2. The Substrate Used in the Invention

Various materials, in shape and composition, can be used as a substratein the present invention. All suitable substrates provide high surfacearea while promoting the conveyance of adsorbates to the adsorbent sitesthat bind them (primarily) by forced convective transport. The media istypically provided packed within a container, such as a column, that isdesigned to hold the media so that it will not be carried away in theflowing blood (a.k.a. media migration) and permit the flow of blood pastessentially all of the media's surface. Useful substrates for creatingthe adsorption media include non-porous rigid beads, particles, orpacking, reticulated foams, a rigid monolithic bed (e.g. formed fromsintered beads or particles), a column packed with woven or non wovenfabric, a column packed with a yarn or solid or hollow dense (notmicroporous) monofilament fibers, a spiral wound cartridge formed fromflat film or dense membrane, or a combination of media such as a mixedbead/fabric cartridge. A suitable substrate for use in the presentinvention is one that is initially microporous but becomes essentiallynonporous when the surface is treated before, during or after thecreation of adsorption sites, e.g., via end-point-attached heparin.

The column has a macroporous structure that presents a high surface areato the blood or serum while preventing a large pressure drop and highshear rates. In addition to the potential for damaging the blood byhemolysis, high pressure drops should be avoided because they can shutdown extracorporeal circuits equipped with automatic shut offs thatrespond to pressure drop.

The substrate may also take the form of a dense a.k.a. barrier membrane.In this embodiment, the surface of a non-porous film is modified bybinding heparin, heparan sulphate or another adsorbent polysaccharidetogether with optional adsorbing groups not derived from heparin,heparan sulphate, or the adsorbent polysaccharide to the membrane'ssurface. Alternatively, a microporous membrane may be rendered nonporousor ‘dense’ before, during or after attachment of binding sites byfilling the pores with essentially non-porous material, e.g., a polymer.The membrane in sheet or (hollow) fiber form may be arranged within ahousing to present high surface area for blood contact that is suitablefor use in the practice of the present invention.

2.1. Beads as Substrate

One useful substrate is in the form of solid beads or particles. The‘beads’ can be made of materials that are sufficiently rigid to resistdeformation/compaction under the encountered flow rates. Resistance todeformation is necessary to maintain the free volume and subsequent lowpressure drop of the packed bed ‘contactor’. The substantial lack ofaccessible pores in the bulk of the substrate eliminates the need foradsorbates to diffuse into the pores prior to adsorption. The adsorptionsites of the present invention are primarily on the surface of the mediaand are thus positioned to be accessible to adsorbates in the blooddelivered to that surface largely by convective transport. Suitablesubstrates need not be perfectly smooth on their surface since roughnessproduces a desirable increase in surface area for attachment of bindingsites, e.g. by covalent or ionic bonding of heparin. Accessible internalpores with molecular dimension, on the other hand, are largely avoidedto eliminate the need for adsorbates to diffuse into the pores beforeattaching to binding sites.

Various kinds of beads can be used in the invention. Useful beads shouldhave sufficient size and rigidity to avoid deformation/compaction duringuse in the method, and have sufficient surface area to be capable ofbeing coated with heparin for use in the method.

Evidence of sufficient substrate rigidity is the absence of asignificant increase in pressure drop across the adsorption bed duringabout one hour of flow of water or saline at rates typical of clinicaluse: for example, <10-50% increase relative to the initial pressure drop(measured within the first minute of flow) when measured at similar flowrate, e.g, of saline.

The beads or other high-surface-area substrates may be made from anumber of different biocompatible materials, such as natural orsynthetic polymers or non-polymeric material including glasses, ceramicsand metals, that are essentially free of leachable impurities. Someexemplary polymers including polyurethane, polymethylmethacrylate,polyethylene or co-polymers of ethylene and other monomers, polyethyleneimine, polypropylene, and polyisobutylene. Examples of useful substratesinclude nonporous Ultra High Molecular Weight PolyEthylene (UHMWPE).Other suitable beads are polystyrene, high density and low densitypolyethylene, silica, polyurethane, and chitosan.

Methods for making such beads are per se known in the art. Polyethylenebeads and other polyolefin beads are produced directly during thesynthesis process and can often be used without further size reduction.Other polymers may need to be ground or spray dried and classified, orotherwise processed to create beads of the desired size distribution andshape.

As noted above, for use in the method of the invention, the size of thechannels or interstitial space between individual beads forextracorporeal blood filtration should be optimized to prevent ahigh-pressure drop between the inlet and outlet of the cartridge, topermit safe passage of the blood cells between the individual beads in ahigh flow environment, and to provide appropriate interstitial surfacearea for binding of the polysaccharide adsorbent to the cytokines orpathogens in the blood. In a close packed bed of 300-micron, roughlyspherical beads, an appropriate interstitial pore size is approximately68 microns in diameter. Useful beads have a size ranging from about 100to above 500 microns in diameter. The average size of the beads can befrom 150 to 450 microns. For example, polyethylene beads from PolymerTechnology Group (Berkeley, USA) having an average diameter of 0.3 mmare suitable. The interstitial pore is a function of bead size.

For use, the suitable beads are housed in a container, such as a column.

Other suitable forms of substrate are described below.

Reticulated foams have open cells and can be made from, for example,polyurethanes and polyethylenes. Control of pore size can be achieved bycontrolling the manufacturing method. In general, reticulated foams canhave between 3 and 100 pores/inch and can exhibit a surface area of ≥66cm²/cm³.

Beads can be sintered into a monolithic porous structure through eitherchemical or physical means. Polyethylene beads can be sintered byheating the beads above their melting temperature in a cartridge andapplying pressure. The resulting interstitial pore size is slightlyreduced from the interstitial pore size of a packed bed of non-sinteredbeads of equal size. This reduction can be determined empirically andused to produce the desired final interstitial pore size.

A column or other housing shape can be packed with either woven ornon-woven heparinized fabric or the heparin, heparan sulphate oroptional non-heparin adsorption sites may be attached, e.g. by covalent,ionic or other chemical or physical bonds, after the housing has beenfilled with the substrate media. By controlling the fiber denier anddensity of the fabric during weaving or knitting or during the creationof a non-woven web, the interstitial pore size can be controlled. Usefulnon-woven fabrics may be in the form of felts, melt-blown, orelectrostatically spun webs, having a random orientation held togetherby entanglement of the fibers and/or adhesion or cohesion ofintersecting fibers. Useful woven fabrics have a more defined andnon-random structure.

A column can be packed with fibers or yarns made from fibers.Polyethylene, and other fibers, can be drawn into thin hollow or solidmonofilament fibers or multifilament yarns, that can be packed intocartridges in the same way that hollow fiber membranes are installedwithin conventional hemodialysis cartridges or blood oxygenators. In thepresent invention originally porous hollow fibers are rendered dense ornon-porous before, during or after binding heparin or other adsorbentsto the outer and/or inner surfaces. Dyneema Purity® from Royal DSM is ahigh-strength solid fiber made of UHMWPE. Dyneema can be heparinized andpacked into a cartridge to provide a high-surface area support for theremoval of cytokines and pathogens.

A spiral wound cartridge contains a thin film or membrane that istightly wound together with optional spacer materials to prevent contactof adjacent surfaces. The membrane can be made from polymers such aspolyurethane, polyethylene polypropylene, polysulfone, polycarbonate,PET, PBT, etc.

2.2. Attachment of the Adsorbant Polysaccharide

The adsorbant polysaccharide of the invention can be bound to thesurface of the solid substrate by various methods, including covalentattachment or ionic attachment.

The adsorption media of the present invention can comprise heparincovalently linked to the surface of the solid substrate. Various per seknown methods can be used to attach heparin to the desired substrate,such as described in a review article by Wendel and Ziemer. (H. P Wendeland G. Ziemer, European Journal of Cardio-thoracic Surgery 16 (1999)342-350). In one embodiment, the heparin is linked to the solidsubstrate by covalent end-point attachment. This method increases thesafety of the device by reducing or eliminating the release of heparinfrom the substrate surface that could enter the blood stream. ‘Leaching’of heparin by and into the blood is to be avoided because it canincrease the risk of bleeding and heparin-induced thrombocytopenia.

Covalent attachment of the polysaccharide, such as heparin, to a solidsubstrate provides better control of parameters such as surface densityand orientation of the immobilized molecules as compared to non-covalentattachment. These parameters have been shown by the inventors to beimportant in order to provide optimal cytokine or pathogen binding tothe immobilized carbohydrate molecules. The surface concentration ofheparin on the solid substrate can be in the range of 1-10 μg/cm².Covalent end-point attachment means that the polysaccharide, such asheparin is covalently attached to the solid substrate via the terminalresidue of the heparin molecule. Heparin can also be bound at multiplepoints. The end-point attachment is preferred.

If beads are used, they may be hydrophilized prior to attachment of thepolysaccharide, such as heparin, or other compound. Possible methods ofpreparing the beads include acid etching, plasma treating, and exposureto strong oxidizers such as potassium permanganate.

2.3. Amount of Polysaccharide/Gram Substrate

The amount of polysaccharide adsorbent per gram substrate can vary. Inone particular embodiment, if beads are used, the amount ofpolysaccharide, such as heparin per gram bead is determined by thenumber of layers used and also the size of the beads. The larger thebead, the less polysaccharide, such as heparin per gram of bead isachieved. One preferred amount is 2.0±0.5 mg heparin/g bead per the MBTHmethod.

The molecular weight of polysaccharide used in the claimed methods canvary. For example, native heparin has an average molecular weight of 22kDa. Nitric acid degraded heparin has a molecular weight of 8 kDa.

3. Mixture of Beads with Different Surface Functionality

Heparin is a biologically active carbohydrate that can bind cytokines,pathogens, and other proteins. But heparin is best known, and mostwidely used as an anticoagulant that prevents blood from clotting. Ithas been safely used clinically for 50 years as an injectable, systemicanticoagulant. In addition, it has been used for many years bymanufacturers as a coating or surface treatment for medical devices forthe sole purpose of improving their safety in blood-contactingapplications. This is particularly important in those devices that mustexpose large surface area to blood for purposes of mass transport.Examples include dialyzers and blood oxygenators. The surface of theadsorption media used in the present invention incorporates heparin fortwo purposes: 1) heparin's ability to bind pathogens, cytokines andother blood-borne substances that contribute to disease, and 2)heparin's ability to prevent blood coagulation and related reactionsupon contact with a foreign, e.g. manmade, surface. Thus heparin is acritical component of the adsorption media of the present inventionbecause it provide both efficacy and safety during the removal ofharmful substances from blood and other biological fluids.

In addition to heparin and heparan sulfate, there are other biologicallyactive chemical moieties including other carbohydrates that can removeharmful substances from blood and biological fluids that are notefficiently removed by immobilized heparin alone. For example, chitosan,a highly cationic, positively-charged carbohydrate will bind endotoxins.Other positively charged molecules, such as polyethylene imine (PEI),can also bind endotoxins. However, cationic surfaces are significantlyless blood compatible than heparinized surfaces and can lead toincreased thrombogenicity, a dangerous condition in blood contactingdevices. (See Sagnella S., and Mai-Ngam K. 2005, Colloids and SurfacesB: Biointerfaces, Vol. 42, pp. 147-155, Chitosan based surfactantpolymers designed to improve blood compatibility on biomaterials andKeuren J. F. W., Wielders S. J. H., Willems G. M., Morra M., Cahalan L.,Cahalan P., and Lindhout T. 2003, Biomaterials, Vol. 24, pp. 1917-1924.Thrombogenecity of polysaccharide-coated surfaces). While it is possibleto use an adsorption cartridge containing PEI, chitosan or otherinherently throbogenic surfaces as a bioactive adsorbent to remove LPSor endotoxins from blood, due to the severe clotting risk, the patientwould need a high dose of systemic anticoagulant. In the case ofsystemic heparin, this could lead to a bleeding risk and possiblethrombocytopenia.

The method of the invention prepares the adsorption bed from a mixtureof heparinized media and media which is inherently thrombogenic. Byassembling an adsorption cartridge with both heparinized surfaces and,for example, cationic surfaces (or other inherently thrombogenicsurfaces), cytokines, pathogens, and endotoxins can all be safelyremoved from blood or biological fluid.

Another biologically active, but thrombogenic carbohydrate is sialicacid. Sialic acid is known to bind virus lectins, including influenza.(C, Mandal. 1990, Experientia, Vol. 46, pp. 433-439, Sialic acid bindingLectins.) A mixed cartridge of heparinized beads and sialic acid coatedbeads can be useful in treating patients, such as during an influenzapandemic.

Abdominal septic shock is usually caused by E. coli which is a gramnegative bacterial. Gram negative bacteria typically do not bind toheparin, and therefore it would be useful to have an adsorption columnwith multifunctionality to bind these bacteria in addition to cytokinesand/or endotoxins. Carbohydrates with mannose sequences, such as methylα-D-mannopyranoside, are known to bind E. coli, K pneumonia, P.aeruginosa, and Salmonella. (Ofek I., and Beachey E. H. 1, 1978,Infection and Immunity, Vol. 22, pp. 247-254, Mannose Binding andEpithelial Cell Adherence of Escherichia coli and Sharon, N. 2, 1987,FEBS letters, Vol. 217, pp. 145-157 Bacterial lectins, cell-cellrecognition and infectious disease.)

The use of this embodiment is based on the concept that anantithrombogenic surface in intimate contact with, or in close proximityto a thrombogenic surface can prevent clinically significant thrombusformation that would otherwise occur if the inherently thrombogenicsurface was used alone. In the case of adsorption media in the formbeads or particles a preferred application of this invention is to blendthe different adsorption media together before packing them into acartridge or other housing. This provides intimate contact among thevarious surface chemistries on adjacent beads while permitting efficientmanufacturing of adsorption cartridges or filters. A related approach isto layer the different media in a ‘parfait-type’ arragnement within thehousing such that the blood contacts the different media in series orparallel flow. One arrangement of the different media within a cartridgeis to position unblended antithrombogenic media at the entrance and/orthe exit of the cartridge, with an optionally blended region containingthe more thrombogenic media interposed between the entrance and exitregions. In the case of media in fiber form, a mixed woven, knitted, ornonwoven structure can be prepared by methods well known in the textileindustry to form fabric from the mixed fiber. Alternatively a yarn canbe prepared from finer multifilament yarn or monfilment made from two ormore fibers with different surface chemistries, as long as one fibertype contains a surface that actively prevents blood clotting oncontact. The mixed-fiber yarn can then be used to prepare fabric forblood contact. Hollow fiber or solid fiber adsorption media can beblended and used to make cartridges that resemble hollow-fiber dialyzersor oxygenators. For membrane or film-type adsorption media of the typethat is used in a spiral-wound adsorption cartridges, two or moresurface chemistries may be used in close proximity to each other suchthat the blood must contact both surface chemistries (nearly)simultaneously. This could be done with a regular or random array of thevarious binding groups within the surface layer of the membrane film, orby forming a flow path for blood between two closely-spaced membranefilms, one of which is antithrobogenic.

4. Device for Use in the Methods of the Invention

Another aspect of the present invention provides use of a devicecomprising the adsorbent modified solid substrate, the adsorbent havinga binding affinity for a cytokine or pathogen, for extracorporealremoval of the cytokine or pathogen from mammalian blood.

A device as referred to in the use and method according to the inventionmay comprise a conventional device for extracorporeal treatment of bloodand serum from patients, e.g. suffering from renal failure.

Local blood flow patterns in blood contacting medical devices forextracorporeal circulation are known to influence clot formation viashear activation and aggregation of platelets in stagnant zones.Consequently, a device as used in the various aspects of the inventionshould be designed in a fashion that does not create these problems.

A device as used in some embodiments of the invention may for examplehave the following properties:

-   -   A blood flow in the range of 150-2000 ml/min, or if measured by        linear flow velocity of ≥8 cm/min.    -   Low flow resistance.    -   Large surface area of substrate having carbohydrates immobilized        thereto, e.g. about 0.1-1 m².    -   Stable coating (no clinically significant leakage of        carbohydrate to the blood in contact therewith).    -   Proper haemodynamic properties in the device (no stagnant        zones).    -   Optimal biocompatibility.

A non-limiting example of such a device, which can be used in a use or amethod according to the present invention, is a pediatric haemoflowdialyzer which is an extracorporeal blood filtration device for removingcytokine molecules and which is compatible with high flow rates. Onesuch device is available from Exthera Medical. Other models or types ofdevices for extracorporeal treatment of blood or serum may also be used,such as the Prisma M10 haemofilter/dialyzer from Gambro AB, Sweden.

High-flow conditions can be defined as blood flow above the diffusionlimit.

5. Cytokines

As used herein, the term “cytokine” means a protein, released forinstance in connection with microbial infection or immunization,selected from the group consisting of interleukins, interferons,chemokines and tumour necrosis factors. Examples of the cytokine(s) arevascular cell adhesion molecule (VCAM), antithrombin, Regulated onActivation Normal T Expressed and Secreted protein (RANTES), interferon,tumor necrosis factor alpha (TNF-alpha), tumor necrosis factor beta(TNF-beta), interleukin-1 (IL-1), IL-8, GRO-α and interleukin-6 (IL-6).

The method also provides for the selective removal of cytokines from theblood by strongly adsorbing heparin-binding molecules. Some moleculeshave a higher binding affinity for heparin than others. For example,TNF-α has a high affinity for heparin.

6. Pathogens

An additional aspect of the invention provides a method of treating adisease by removing cytokines and/or pathogens from mammalian blood bycontacting mammalian blood with the solid substrate disclosed in themethod above. Examples of pathogens that can be removed from the bloodusing heparinized substrate according to the invention include:

Viruses—Adenovirus, Coronavirus, Dengue virus, Hepatitis B, Hepatitis C,HIV, HPV Cytomegalovirus, and others.

Bacteria—Bacillus anthracis, Chlamydia pneumoniaem, Listeriamonocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, MRSA,Streptococcus pyrogenes, Yersinia enterocolitica, and others

Parasites—Giardia lambitia, plasmodium spp. and others.

See also, Chen, Y. Gotte M., Liu J., and Park P. W., Mol. Cells, 26,415-426.

One example of a disease to be treated according to the invention issepsis. Sepsis is generally considered to be a systemic response to aninfection which can lead to organ failure and often death. The conditioncan be triggered by a bacterial, viral, parasitic or fungal infection.The condition is known to be particularly dangerous in hospitals wherepatients may already be immuno-compromised. During sepsis, the patientexperiences a so-called cytokine storm and the body's immune systemattacks healthy tissue that leads to multiple organ failure in highlyperfused organs. Reducing TNF-α and other inflammatory molecules willmodulate the immune response and could act as an organ preservationstrategy. Additionally, any heparin-binding pathogens in the blood canbe removed which would help reduce further colonization and could reducethe amount of antibiotics needed to treat an infection. This couldimprove patient safety by reducing side effect risks associated withantibiotic therapy.

The methods of the present invention can be employed either before orafter other conventional treatments, such as administration ofantibiotics.

7. Combining the Inventions with Additional Filtration/Separation Steps

In an embodiment of the treatment method according to the presentinvention, the extraction and reintroduction of blood may be performedin a continuous loop, which loop comprises a part of the bloodstream ofthe subject.

In a further aspect the methods described above can be combined withother methods to filter or treat mammalian blood. For example, acartridge that is based on convection kinetics can then be used inseries with conventional extracorporeal circuits such as CPB,hemodialysis, and oxygenation.

8. Examples

The various aspects of the invention are further described in thefollowing examples. These examples are not intended to be limiting. Forinstance, in the present examples heparin is used. However, othercarbohydrates and polysaccharide adsorbents may be used alone or inaddition to the heparin-coated substrates exemplified below.

8.1. Example 1

Preparation of Heparin Column

Polyethylene (PE) beads, with an average diameter of 0.3 mm (lot no.180153), are supplied by the Polymer Technology Group (Berkeley, USA)and the columns (Mobicol, 1 mL) are obtained from MoBiTec (Germany).Heparin and polyethyleneimine (PEI) are purchased from ScientificProtein Laboratories (Waunakee, Wis., USA) and BASF (Ludwigshafen,Germany) respectively. All chemicals used are of analytical grade orbetter.

Immobilization of heparin onto the beads was performed as described byLarm et al. (Larm O, Larsson R, Olsson P. A new non-thrombogenic surfaceprepared by selective covalent binding of heparin via a modifiedreducing terminal residue. Biomater Med Devices Artif Organs 1983; 11:161-173).

The polymeric surface was heparinized using the general proceduredescribed below.

The polymeric surface is etched with a oxidizing agent (potassiumpermanganate, ammoniumperoxidisulfate) in order to introduce hydrophiliccharacteristics together with some reactive functional groups (—SO₃H,—OH, —C═O, —C═C—). The surface can also be etched with plasma or corona.For example, the PE-beads are etched with an oxidizing agent (potassiumpermanganate in sulphuric acid). These hydrophilized beads, inter aliacontaining OH-groups and double bonds, are later used as controls.

Reactive amino functions are introduced by treatment with a polyamine,polyethylenimine (PEI) or chitosan. For some purposes the polyamines maybe stabilized on the surface by cross linking with bifunctionalreagents, such as crotonaldehyde or glutaraldehyde.

The coating is further stabilized by ionic cross linking with a sulfatedpolysaccharide (dextran sulfate or heparin). If necessary these stepsare repeated and a sandwich structure is built up. Careful rinsing(water, suitable buffers) should be performed between each step. After alast addition of PEI or chitosan, end-point attachment (EPA) to theaminated surface of native heparin is done by reductive amination,utilizing the aldehyde function in the reducing terminal residue innative heparin.

A more reactive aldehyde function in the reducing terminal residue canbe achieved by partial, nitrous degradation of heparin. This shortensthe reaction time, but the immobilized heparin will have a lowermolecular weight. The coupling is performed in aqueous solution, byreductive amination (cyanoborohydride, CNBH₃ ⁻).

In this alternate method, the aminated media is suspended in acetatebuffer (800 ml, 0.1 M, pH 4.0) and 4.0 g nitrous acid degraded heparin(heparin from Pharmacia, Sweden) was added. After shaking for 0.5 h,NaBH₃CN (0.4 g) was added. The reaction mixture was shaken for 24 h andthen processed as above, yielding heparinized media.

1-10 μg/cm² of heparin can be coupled to all hydrophilic surfaces likeglass, cellulose, chitin etc, and more or less all hydrophobic polymerslike polyvinyl chloride, polyethylene, polycarbonate, polystyrene, PTFEetc.

The resulting PE-beads, with covalently end-point attached heparin, aresterilized with ethylenoxide (ETO) and rinsed with 0.9% sodium chlorideand ultra pure water. The amount heparin was determined to be 2.0 mgheparin/g bead with the MBTH method. (Larm O, Larsson R, Olsson P. A newnon-thrombogenic surface prepared by selective covalent binding ofheparin via a modified reducing terminal residue. Biomater Med DevicesArtif Organs 1983; 11: 161-173 and Riesenfeld J, Roden L. Quantitativeanalysis of N-sulfated, N-acetylated, and unsubstituted glucosamineamino groups in heparin and related polysaccharides. Anal Biochem 1990;188: 383-389).

The polyethylene beads that are used had a mean diameter of 0.3 mm andare heparinized with a technology that guaranteed that the heparinmolecules are covalently end point attached to the surface, therebymaking the carbohydrate chains more accessible for proteins withaffinity for heparin/heparan sulphate. The mean molecular weight of theimmobilized heparin was about 8 kDa, while 2 mg (equal to approximately360 IU) was coupled to each gram of beads. The integrity of this surfacewas verified by the expected removal of 75% of antithrombin (AT)concentrations from the blood passed over heparinized, but notnon-heparinized, beads.

These data corresponds well with the previous observations fromextracorporeal lung assistance (ECLA) on septic patients using surfaceheparinized oxygenators published by Bindslev et al. (Bindslev L, EklundJ, Norlander O, Swedenborg J, et al. Treatment of acute respiratoryfailure by extracorporeal carbon dioxide elimination performed with asurface heparinized artificial lung. Anesthesiology 1987; 67: 117-120.)

8.2. Example 2

Patients

The study protocol was approved by the local ethics committee at theKarolinska University Hospital and signed informed consent was obtainedfrom each patient. Arterial blood was drawn from the hemodialyzers ofthree septic (fever >38° C., chills, leukocytes >12×10⁹ cells/L)patients (2M/1F, aged 43, 56 and 68 years; Table 1).

TABLE 1 Clinical characteristics of patients donating blood. White Meanblood Body arterial Respiratory cell Age temperature Heart rate pressurerate count Patient Sex (years) (° C.) (beats/min) (mmHg) (breaths/min)(10⁹/L) 1 M 43 39.2° 110 76 20 19.0 2 M 56 38.6° 90 95 18 17.5 3 F 6838.5° 100 89 21 19.5

The patients are previously administered with broad-spectrum antibiotics(ceftazidime or cefuroxime together with an aminoglycoside; one dose ofeach) and heparin (200 IU/kg body weight at the start of the dialysis).The blood was collected in EDTA vacuum tubes and immediately transferredto an adjacent room where 1 mL was applied to the previously preparedcolumns and passed through using a roller-pump at one of 1, 5 and 10mL/min. Blood that had passed through the columns was immediatelycollected at the other end and cold-centrifuged (4500 G). Thesupernatants are subsequently collected and frozen at −80° C. for lateranalysis.

8.3. Example 3

Quantitative Analysis of Cytokines

The amounts of cytokines are determined using photoluminescence with aplate reader (Multiskan Ascent). Each sample was measured in threewells, and the geometric mean used for analysis. The intraassaycoefficient of variation was below 8% for all kits. We used Coamaticantithrombin kit (Haemochrom, cat #8211991), Quantikine Human IL-6 (R&DSystems, cat #D6050), Quantikine Human IL-10 (R&D Systems, cat #D1000B),Protein C Antigen Test 96 (HL Scandinavia AB, roduct #H5285), HumanCCL5/RANTES Quantikine (R&D Systems, cat #DRN00B), Quantikine HumansVCAM-1 (CD106) (R&D Systems, cat #DVC00), Quantikine Human IFN-gamma(R&D Systems, cat #DIF50), Quantikine HS TNF-alpha/TNFSF1A (R&D Systems,cat #HSTA00D) and BD OptEIA Human C5a ELISA Kit II (BD Biosciences, cat#557965).

Statistical Evaluation

Paired Kruskal-Wallis test was used to compare the before and aftercolumn blood concentrations of each cytokine, with a two-tailed p-valuebelow 0.05 indicating significance. The results are summarized in Table2.

TABLE 2 Concentrations of measured cytokines before and after bloodpassage through different columns. Cytokine Control beads Heparinizedbeads Before After After passage passage p-value passage p-value 10 gbeads/1 mL blood VCAM (ng/mL) 115.5 99.0 0.30 88.8 <0.05 (−14%) (−23%)IL-6 (pg/mL) 19.7 17.4 0.17 15.0 0.16 (−12%) (−24%) RANTES (pg/mL) 147.1832.5  <0.05 156.4  0.61 (+466%)   (+6%) Interferon-g 340.0 296.0  0.32287.0  0.45 (pg/mL) (−13%) (−16%) TNF-α (pg/mL) 50.3 45.6 0.46 20.6<0.01  (−9%) (−59%) Antithrombin 105.0 92.5 0.10 26.0 <0.01 (% activity)1 g beads/1 mL blood VCAM (ng/mL) 115.5 99.5 0.30 89.2 <0.05 (−14%)(−23%) IL-6 (pg/mL) 19.7 17.5 0.17 15.9 0.16 (−11%) (−19%) RANTES(pg/mL) 147.1 833.0  <0.05 156.8  0.61 (+466%)   (+7%) Interferon-g340.0 296.7  0.32 287.5  0.45 (pg/mL) (−13%) (−15%) TNF-α (pg/mL) 50.346.3 0.46 21.1 <0.01  (−8%) (−58%) Antithrombin 105.0 93.0 0.10 26.8<0.01 (% activity)Cytokine Binding

Pre- and post-column concentrations of analyzed cytokines are shown inTable 2 and FIG. 1. Briefly, passage through the heparinized beadsresulted in a significantly bigger decrease in blood VCAM and TNF ascompared to non-heparinized beads.

Impact of Bead Volume

Data obtained with a 1:1 and 1:10 blood-to-bead volume did not varysignificantly (Table 2).

Impact of Flow Rate

Varying the blood flow rate from 1 up to 10 mL/min did not significantlyaffect the amount removed of the respective cytokines, indicating thatthe observed binding to the immobilized heparin molecules is a veryrapid event and is clearly not dependent on diffusion kinetics.

8.4. Example 4

In this example, 5 liters of platelet poor plasma in which recombinantTNF-α had been added was tested with a clinically sized cartridge. A 300ml cartridge was packed with heparinized PE beads as described inExample 1. The device was sterilized using ETO sterilization using the16 hour cycle with a temperature of 50 degrees. The device was allowedto “gas out” for an additional 12 hours prior to the start of the study.

5.1 L of frozen, unfiltered porcine heparinized platelet poor plasmafrom Innovative Research was purchased, and stored in a −20 degreefreezer until the day of use. 1 mg of Recombinant Human Tumor NecrosisFactor-α was received from Invitrogen in powder form.

The morning of the procedure the plasma was removed from the −20 degreeC. freezer and placed into a warm water bath to thaw. 1 ml sterile waterwas mixed with the 1 mg powdered TNF-α to reconstitute to aconcentration of 1 mg/ml.

A Fresenius 2008K dialysis machine was set up with a “Combiset”hemodyalisis blood tubing (standard tubing used on the Freseniusmachine) in a closed system set up with the seraph hemofilter in placeof the Fresenius Kidney. 5 L of plasma was transferred into a 5 Lreservoir bag which was connected to the arterial and venous patientlines. The dialysis machine along with tubing was primed with saline toensure proper function and that there was no air through the closedcircuit.

The 5 L bag of plasma was placed on a plate rocker with a Bair Huggerwarming blanket wrapped around to maintain temperature throughout theprocedure. At this point the pre-infusion control sample was collectedand placed in liquid nitrogen for snap freezing. The sample was thenmoved to a sample storage box and placed on dry ice. After allconnections were confirmed, 0.415 ml TNF-α was injected into the port ofthe reservoir bag. The plasma with TNF-α was allowed to mix on therocker for 10 minutes before the post-infusion control sample wascollected. The post-infusion sample was collected, snap frozen in liquidnitrogen, then moved to the sample box on dry ice. The dialysis systemwas purged of saline and the system clock was started when the plasmawas passed through the closed system. The first sample was collectedupstream from the filter at 5 minutes.

For the 1^(st) hour of the test run, samples were collected every 5minutes from the port immediately upstream and also immediatelydownstream from the Hemofilter. The samples were snap frozen in liquidnitrogen and placed in the sample storage box on dry ice.

For the 2^(nd) and 3^(rd) hour of the test run the samples werecollected every 10 minutes from the port immediately upstream andimmediately downstream from the hemofilter. The samples were snap frozenin liquid nitrogen and placed in the sample storage box on dry ice.

For the 4^(th) and 5^(th) hour of the test run, samples were collectedevery 20 minutes from both the upstream and downstream ports. Thesesamples were also snap frozen in liquid nitrogen, and placed in thesample storage box on dry ice.

At each sample collection, both upstream and downstream for each timepoint, a new needle and a new syringe was used to avoid residual TNF-α.

ELISAs were performed to monitor the amount of TNF-α in the plasma. Tocoat the 96-well plates, 10 μL of the capture antibody was diluted in 10μL of coating buffer A and 100 μL was added to each well. The plate wascovered with parafilm and stored at 4° C. overnight or over the weekend.Samples were removed from the −80° C. freezer, logged, and allowed tothaw. Plates were removed from the 4° C. refrigerator and wellsaspirated, washed once with 400 μL/well of Assay buffer, then invertedand blotted on absorbent paper towels. 300 μL of Assay buffer were addedto each well and the plates were incubated for 60 minutes. During thistime, standards were prepared from the TNF-α standard included with theCytoSet by dilution in Assay buffer. Thawed samples were diluted inassay buffer. Once samples were diluted, they were replaced in theproper places in the freezer boxes and returned to the −80° C. freezer,as indicated by the log. Plates were washed again with 400 μL/well ofAssay buffer, inverted, and blotted. The 100 μL was placed in the wellsaccording to the ELISA template sheets. Samples and standards were runin duplicate and a standard curve was included on each plate. 8.8 μL ofdetection antibody was diluted in 5.49 mL Assay buffer and 50 μL wasadded to each well as soon as all samples and standards were added.Plates were incubated for 2 hours at room temperature on an orbitalshaker. Plates were washed and aspirated 5 times as described above. Thestreptavidin conjugate (16 μL) was diluted in 10 mL of Assay buffer and100 mL was added to each well. Plates were incubated 30 minutes at roomtemperature on the orbital shaker then washed with Assay buffer 5 timesas described above. 100 μL of the TMB solution was added to each welland plates were incubated a further 30 minutes on the orbital shaker.100 μL of the stop solution was added to each well and the plate wasread at 450 nm on a Vmax plate reader within 30 minutes of adding thestop solution.

The results of the test are plotted in FIG. 1. Within 40 minutes ofcontinuous cycling of TNF rich serum, an 80% reduction of TNF wasobserved, with no subsequent release. With ideal mixing, all of the TNFin the plasma would have passed over the adsorption column in 33minutes. Removal of TNF-α through adsorption based on convectionkinetics is demonstrated as the majority of TNF was captured in thistime period.

8.5. Example 5

A quartz crystal microbalance (QCM) experiment was performed to attemptto normalize the uncompetitive binding capacity of TNF-α to aheparinized surface. QCM is a technique that can detect the weight ofadsorbents on specially prepared crystals. The minimum detection limitis 0.5 ng/cm². QCM detects the resonance frequency of the crystal. Asthe mass of the crystal increases through adsorption, the resonancefrequency changes in proportion to the gain in mass. The change in mass(Δm) is described in the following equation

${\Delta\; m} = {{- C}\;\frac{1}{n}\Delta\; f\mspace{14mu}\begin{matrix}{{C = 17},{7{ng}\;{cm}^{- 2}s^{- 1}}} \\{n - {overtone}}\end{matrix}}$where Δf is the change in frequency.

Polystyrene coated QCM crystals were heparinized following the methoddescribed in example 1. A 120 ml solution was prepared where recombinantTNF-α was added to PBS for a final concentration of 83 μg/L. Thesolution was then flowed through a cell containing 4 QCM crystals at arate of 50 μl/min. Two crystals were heparinized and two were controlcrystals (untreated polystyrene). The total time of the experiment was20 hours. The results for the amount of TNF-α adsorbed on theheparinized crystals and the controls are shown in FIG. 2. A maximum of1234 ng/cm² of TNF-α adsorbed on the heparinized crystal and aadsorption on the control surfaces was negligible.

8.6. Example 6

A study was performed testing the removal of cytokines from plasmasampled from Bacillus anthracis infected macaques using heparinizedbeads. The plasma was collected at the time of death of several animalsand pooled. 1 ml filter syringes were packed with either 0.5 grams of PEbeads heparinized following the procedure outlined in Example 1 oruntreated PE beads for use as controls. A total of three sample syringesand three control syringes were used. A top porous plate was placed ontop of the beads to keep the beads from floating as saline or plasma wasadded to the filter syringes. The filter syringes were primed usingusing 2 ml of Tris-buffered Saline (TBS). The 2 ml of saline was passedthrough the filter syringe using a 5 ml syringe. The plunger of the 5 mlsyringe was then retracted and filled with 4 ml of air. The air was thenpassed through the filter syringe to force the remaining saline out ofthe beads. 0.5 ml of plasma was then pulled into the 5 ml syringe andthen pressed through the filter syringe. An additional 4 ml of air waspassed through the filter syringe to remove any residual plasma. Afterthe plasma drained through the syringe, aliquots were sampled and flashfroze for cytokine concentration analysis. A Luminex® Multiplex assaywas used to test for GRO-α, IL-8, MIP-1β, Rantes, and TNF-β. The resultsare summarized in Table 3.

TABLE 3 Passage Passage Before Through Through Passage Control Heparin %Removed Levels Beads % Removed Beads Heparin Cytokine (pg/ml) (pg/ml)Control (pg/ml) Beads GRO-α 263.5 158.5 39.8% 77.66 70.6% IL-8 504.9460.8 8.7% 312.4 38.1% MIP-1β 66.2 56.4 14.8% 51.8 21.8% RANTES 1105.3930.3 15.8% 559.7 49.4% TNF-β 7.3 7.9 −8.2% 4.3 41.4%

8.7. Example 7

Cartridge to Bind Cytokines and Endotoxins Using a Layered Assembly

A cartridge is built using two different adsorption media. Heparinizedbeads are used to capture cytokines and PEI beads are used to captureendotoxins. By controlling the ratio of heparinized to PEI beads, bloodcompatibility can be maintained. The PEI beads are made following thefirst two steps in Example 1.

In this example, a cartridge is built with heparinized beads andpolyethyleneimine (PEI) coated beads. A 300 ml adsorption column isfixed to a vertical stand. 50 ml of 300 micron average heparinized beadsare then added to the cartridge and allowed to settle. A 50:50 mixtureof PEI and heparinized beads is then added and makes up the next 200 ml.The cartridge is then filled to the top with a final 50 ml ofheparinized beads. The cartridge is then sealed and the close packing ofthe beads maintain the layered structure of the adsorption media. Inthis cartridge, ⅔^(rds) of the beads are heparinized while ⅓^(rd) isaminated and therefore inherently thrombogenic.

8.8. Example 8

Cartridge to Bind Cytokines and Endotoxins Using a Uniform Mixture ofBeads

A cartridge is built using two different adsorption media. Heparinizedbeads are used to capture cytokines and PEI beads are used to captureendotoxins. By controlling the ratio of heparinized to PEI beads, theoverall blood compatibility of the device can be maintained.

In this example, a cartridge is built with heparinized beads and PEIcoated beads. A 300 ml adsorption column is fixed to a vertical stand.100 ml of PEI coated beads are added to 200 ml of heparinized beads andmixed thoroughly. The 300 ml of beads are then added to the cartridgeand sealed. The close packing of the beads maintains the random mixtureof beads. In this cartridge, ⅔^(rds) of the beads are heparinized while⅓^(rd) are aminated.

8.9. Example 9

Cartridge to Bind Cytokines, Gram Negative Bacteria, and EndotoxinsUsing a Layered Assembly

A cartridge is built using three different adsorption media. Heparinizedbeads are used to capture cytokines, mannose functionalized beads areused to capture gram negative bacteria, and PEI beads are used tocapture endotoxins. By controlling the ratio of heparinized beads,mannose functionalized beads, and PEI coated beads, blood compatibilitycan be maintained.

A 300 ml adsorption column is fixed to a vertical stand. 50 ml of 300micron average heparinized beads are then added to the cartridge andallowed to settle. 200 ml of beads, with equal quantities of heparinizedbeads, PEI beads, and mannose functionalized beads are then added to thecartridge. The cartridge is then filled to the top with a final 50 ml ofheparnized beads. The cartridge is then sealed and the close packing ofthe beads maintain the layered structure of the adsorption media. Inthis cartridge, 55.6% of the beads are heparinized, 22.2% are PEI beads,and 22.2% are mannose functionalized.

The embodiments of the present invention are further described in theclaims below.

We claim:
 1. A cartridge for extracorporeal removal of at least oneadsorbate, the cartridge comprising: a first media being a heparinizedmedia; and a second media, which is cationic and more thrombogenic thanthe first media.
 2. The cartridge of claim 1, wherein the first mediaand the second media are arranged in a layered approach.
 3. Thecartridge of claim 1, wherein the first media and the second media areblended.
 4. The cartridge of claim 3, wherein the blend is random. 5.The cartridge of claim 1, wherein the first media and the second mediaare arranged in a parfait fashion.
 6. The cartridge of claim 1, whereinthe first media are beads comprising a polymer of polyethylene.
 7. Thecartridge of claim 6, wherein the beads have a diameter ranging from 100and 450 microns.
 8. The cartridge of claim 7, wherein the beads have anaverage diameter of 300 microns.
 9. The cartridge of claim 1, whereinthe beads are coated with 0.5 mg to 10 mg heparin per gram of bead. 10.The cartridge of claim 9, wherein the heparin is attached to the bead bycovalent end-point attachment.
 11. The cartridge of claim 1, wherein thesecond media are beads.
 12. The cartridge of claim 11, wherein the beadsare coated with a member selected from the group consisting of PEI,chitosan or sialic acid.
 13. The cartridge of claim 1, wherein the firstmedia and the second media are present in a ratio of 1:1.
 14. Thecartridge of claim 1, wherein the first media and the second media arepresent in a ratio of 2:1.
 15. The cartridge of claim 1, wherein thecartridge comprises a third media.
 16. The cartridge of claim 15,wherein the third media is mannose functionalized beads.
 17. Thecartridge of claim 1, wherein the cartridge is adapted to be used inseries with extracorporeal circuits.
 18. A cartridge adapted forextracorporeal blood circuit, the cartridge comprising: a first mediabeing a heparinized media; and a second media, which is cationic andmore thrombogenic than the first media.